For many years, West Nile virus (WN) has been recognized as one of the most widely distributed flavivirus with geographic range including Africa, the Middle East, Europe and Australia. During 1999, WN virus first established itself in the Northeast and Mid-Atlantic States of the USA and more recently, this virus extended its range to include the southern and mid-western states. Because of this unexpected WN spread from Northeast to South of the USA, this virus is considered a significant re-emerging disease threat and it become a public health problem. Currently, a licensed human vaccine is not available to prevent WN virus disease. Mosquito control is the only practical strategy to combat the spread of disease; however, effective spraying in an attempt to eliminate mosquitoes is difficult to perform in urban areas. Clearly, an effective vaccine is needed to protect at-risk population. For these reasons, we are interested in developing a vaccine against WN. We constructed viable WN/DEN4 chimeras in which the structural preM and E protein genes of the WN virus were substituted for the corresponding genes of dengue type 4 virus (DEN4). We also generated a WN/DEN4 chimera with a 30-nucleotide deletion in the 3'-noncoding region. Both chimeric (WN/DEN4 and its mutant) and parental (WN strain NY99 and DEN4 strain 814669) viruses were evaluated in mice for neurovirulence. Wild-type WN NY99 was highly neurovirulent with an intracerebral (IC) LD50 of 0.35 focus forming units (FFU) in suckling Swiss mice. Wild-type DEN4 that also was prepared on Vero cells was less neurovirulent with an IC LD50 of 407 FFU. Both chimeric WN/DEN4 and WN/DEN4-D30 virus exhibited a significant reduction in neurovirulence compared with its WN parent. All mice that were inoculated IC with a dose of 1, 10, 100, or 1000 FFU of WN/DEN4 or its 3' deletion mutant survived during a 21 day observation period. Only 4 of 11 mice died when inoculated IC with 10,000 FFU of WN/DEN4 chimera. So, the chimera in suckling mice was at least 10,000 times less virulent than its WN parent. These observations are consistent with earlier findings that chimerization of TBEV or LGT with DEN4 significantly reduced its neurovirulence for mice. Peripheral neurovirulence of chimeric and parental viruses was evaluated in 3-week-old Swiss and SCID mice inoculated IP with decimal dilutions of virus ranging from 0.001 to 100,000 FFU. Both chimeras were highly attenuated compared to their WN parent. Notably, IP inoculation of 10,000 FFU of the deletion mutant chimera or 100,000 FFU of the unmodified chimera did not induce fatal encephalitis in 3-week-old Swiss mice, whereas the IP LD50 for the WN parent was 10 FFU. Also, despite the high peripheral virulence of wild-type WN strain NY99 for SCID mice (IP LD50 of 6 FFU), chimerization of WN with DEN4 completely ablated this property of its WN parent for immunodeficient mice. Thus, 3-week-old Swiss or SCID mice survived IP inoculation of 10,000 or 100,000 FFU of chimeric virus. Our observations are consistent with earlier findings that a similar large reduction of peripheral neurovirulence of TBEV or LGT occurs following chimerization with DEN4 . Although highly attenuated, the WN/DEN4 chimeras stimulated a moderate to high level of serum neutralizing antibodies against WN virus. The immune response of mice inoculated with the chimeras was dose-dependent and indicated that the unmodified WN/DEN4 chimera was slightly more immunogenic than the corresponding 3'-deletion mutant. There was a strong correlation between the level of neutralizing antibodies to WN induced by immunization and resistance to subsequent lethal WN challenge. However, 90 to 100% protection against WN challenge was achieved when a single 1000 FFU dose of WN/DEN4 chimera or its 3'-deletion mutant was used for immunization. A higher dose (10,000 FFU) of either chimera provided complete protection to WN challenge. Thus, the WN preM and E proteins of the chimeric viruses represent effective antigens able to induce complete protection to challenge with highly virulent WN. Our observations concerning safety, immunogenicity, and protective efficacy of the chimeric WN/DEN4 vaccine candidates in mice provide a basis for extending our evaluation of the vaccine candidates to non-human primates and domestic animals, such as horses, which are at high risk.